Stem cells, defined by their ability to self-renew and generate mature cells via differentiation, are critical for the development and maintenance of multiple tissues. Stem cells are regulated by cell intrinsic and extrinsic mechanisms that influence transcription, signal transduction, fate determination, and cell cycle control. Stem cell self-renewal mechanisms must be integrated with metabolism, but little is known about how. In addition, it is unclear whether metabolic regulation differs in stem cells compared to other cell types. Hematopoietic stem cells (HSCs) give rise to cells of the blood and immune system throughout life. Investigation of HSC self-renewal mechanisms provides insight into hematopoiesis, leukemogenesis, and basic stem cell and cancer cell biology. In addition, studies on hematopoietic cell types enable improvements to be made in the safety and efficacy of bone marrow transplantation and contribute to the identification of strategies for increasing regenerative capacity of bone marrow. The long term objective of this study is to understand how metabolism integrates with other stem cell self-renewal mechanisms to regulate HSC function. The hypothesis of this proposal is that proline dehydrogenase (Prodh), a mitochondrial metabolic enzyme catalyzing the first and rate-limiting step in proline catabolism, contributes to HSC function through the regulation of cellular energy metabolism and gene expression. Prodh is preferentially expressed in HSCs compared to whole bone marrow cells, suggesting an important or distinct function in HSCs over other hematopoietic cells. In other cell types, Prodh regulates cell survival, proliferation and death, in addition to influencing levels of -ketoglutarate, a metabolite mediating cellular changes in energy metabolism and gene expression. Finally, stem cells are regulated, in part, through the microenvironment, and HSCs reside in a hypoxic microenvironment. In cancer cells grown under hypoxic conditions, Prodh gene expression is upregulated to support cell survival. Together, these ideas are in support of a role for Prodh in HSC function; however, almost nothing is known about the function of Prodh and proline metabolism in stem cell self-renewal or hematopoiesis. In this proposal, the role of proline metabolism in HSC function is investigated by examining the hematopoietic system upon deletion of Prodh from the Mx1-Cre; Prodhfl/fl adult conditional deletion mouse. How Prodh deficiency affects HSC frequency and bone marrow reconstitution capacity will be tested, in addition to effects on HSC and progenitor cell energy production, mitochondrial function and HIF-1 activity. How Prodh deficiency alters the HSC epigenetic state will be assessed by performing gene expression microarray analysis and assays to detect alterations in chromatin structure. Outcomes of the proposed studies will indicate whether HSC metabolism differs from other hematopoietic cells and provide new insight into mechanisms regulating HSC self-renewal.